Bacteriophage is a kind of virus-like microorganism infecting bacteria and generally called ‘phage’ in short. Bacteriophage is an organism having a simple structure wherein a central genetic material composed of nucleic acid is covered by a protein envelope. The nucleic acid is single stranded or double stranded DNA or RNA. To survive, bacteriophage needs a host bacterium and every bacterium has a specific partner bacteriophage. When bacteriophage invades into a host bacterium, it multiplicates itself and then induces expressions of enzymes involved in the decomposition of cell wall of the host bacterium. The enzymes destroy cell wall by attacking the peptidoglycan layer which is responsible for rigidity and mechanical strength of cell wall.
Bacteriophage was first found by Twort, an English bacteriologist, in 1915 during his research on the phenomenon that micrococcus colony is decomposed turning transparent by something. And in 1917, a French bacteriologist d'Herelle found out that there was something that decomposes Shigella disentriae in filtrate of feces of a patient with dysentery, and he continued to study to identify the material, leading to the finding of bacteriophage which means “eating bacteria”. Since then, bacteriophages against Shigella dysenteriae, Salmonella typhi, and Vibrio cholerae were further identified. Since penicillin was found by Flemming in 1950, antibiotics have been widely used and the study on bacteriophage continued only in some East European countries and it became out of concern in many other countries. However, since 2000, multidrug-resistant pathogenic bacteria resulted from over-use and/or mis-use of antibiotics have been frequently reported. Because of potential as an alternative for the conventional antibiotics, bacteriophage became in the spotlight again and the studies on bacteriophage are actively undergoing led by advanced countries.
Even though antibiotics (or antibacterial agents) are still major therapeutic agents for the treatment of various infectious diseases, it has been a serious problem since 1980s that the excessive use of such antibiotics generates numbers of multi-drug resistant strains. In 1986, Staphylococcus aureus having resistance against vancomycin, which is so called ‘the drug of last resort’, and other multi-drug resistant strains were found, giving a great shock to those in medical field. Vancomycin resistant enterococci (VRE) were first reported in France in 1986 and first separated in USA in 1988. Since then, the cases of VRE infection have been increased every year with high frequency, everywhere including Europe, USA, Singapore, Japan, Australia, Korea, etc, making the VRE as a causal agent of nosocomial infections. In Korea, VRE was first isolated in 1992. As for Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus (VRSA) was first found in the early 1990s and was first found in Korea in June, 1996.
Therefore, it is an urgent request to develop a novel antibiotic to treat the infectious diseases caused by bacteria resistant against conventional antibiotics and further to lead national health and medical techniques. Again, it is urgently required to develop an alternative antibiotic to solve the problems of multi-drug resistant bacteria along with the abuse or misuse of the conventional antibiotics and the bio-accumulation of antibiotics. To solve the problem of such resistant bacteria, an alternative antibiotic has to be developed by a completely and fundamentally different method.
The present inventors isolated novel bacteriophage capable of killing specifically Staphylococcus aureus, and deposited the bacteriophage at Korean Agricultural Culture Collection, National Institute of Agricultural Biotechnology on Jun. 14, 2006 (Accession No: KACC 97001P) and at Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology on Jul. 18, 2007 (Accession No: KCTC 11153BP). The related matters have been applied for a patent (Korean Patent Application No. 2006-55461). The present inventors continued the study and as a result isolated another effective bacteriophage, and then deposited the isolated bacteriophage at Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology on Jul. 18, 2007 (Accession No: KCTC 11154BP).
Even if the said two bacteriophages are effective in prevention and treatment of infectious disease caused by Staphylococcus aureus, they still have a few disadvantages. Direct application of bacteriophage, which means the bacteriophage itself is directly used, raises vague aversion, leading to the limitation in use. In addition, to obtain bacteriophage for direct use massively, it is important and necessary to culture host pathogenic bacteria, indicating that there is a high chance of exposure of a worker on pathogenic bacteria. So, a very strict pathogenic bacteria regulation is required. Accordingly it is required to develop a novel substance having characteristics of bacteriophage and capable of killing Staphylococcus aureus in safer way and facilitating wider application.
The present inventors applied for a patent describing a novel antibacterial protein originated from the bacteriophage capable of killing specifically Staphylococcus aureus based on the genetic information thereon (Korean Patent Application No. 2006-73562). It was demonstrated that lytic protein had same lytic effect as that of an endogenous lytic protein in a host when it is extracellularly treated and has a broader bactericidal activity compared to the corresponding bacteriophage itself.
However, like bacteriophage, such antimicrobial proteins take different bacteria as their targets and are different in their antimicrobial spectrum. Thus, it is required to obtain in variety of antimicrobial proteins.
As described hereinbefore, lytic protein (antibacterial protein) derived from bacteriophage is a protein that destroys cell wall of a host bacterium when the bacteriophage comes out of the host bacterium. Such lytic protein derived from bacteriophage is generally called lysin. The lytic protein, lysin, is composed of N-terminal catalytic domain and C-terminal binding domain and these two domains are linked by a short linker. Lysin can have two different catalytic domains, which is a rare case, though. C-terminal binding domain is conjugated with cell wall of target bacteria. The catalytic regions of lysin are conserved when they are in the same class according to Linne's hierarchical classification system but binding domains are different. Such variability of binding domain makes difference in bacteriolytic effect among lytic proteins.
So, preparing an additional lytic protein as described in this invention paves the way to cope with more Staphylococcus aureus and a cocktail of those lytic proteins is expected to bring broader antimicrobial effect, compared with a single lytic protein.